1. Field of Endeavor
The present invention relates to droplet sorting and more particularly to a system for passive sorting of microdroplets in a microfluidic system.
2. State of Technology
United States Published Patent Application No. 2008/0053205 for droplet-based particle sorting provides the following state of technology information: “The present invention relates to droplet-based particle sorting. According to one embodiment, a droplet microactuator is provided and comprises: (a) a suspension of particles; and (b) electrodes arranged for conducting droplet operations using droplets comprising particles. According to another embodiment, a droplet microactuator is provided and comprises a droplet comprising a single particle in the droplet. According to yet another embodiment, a method of transporting a particle is provided, wherein the method comprises providing a droplet comprising the particle and transporting the droplet on a droplet microactuator. According to a still further embodiment, a method of providing a droplet comprising a single particle is provided, wherein the method comprises: (a) providing a droplet comprising suspension of particles; (b) dispensing a droplet from the droplet of (a) to provide a dispensed droplet; and (c) determining whether the dispensed droplet comprises a single particle and/or a desired particle type.”
U.S. Pat. No. 6,941,005 for monitoring and control of droplet sorting provides the following state of technology information:
A. Flow Cell Chamber
One component of the system of the present invention, useful in the method of the present invention is a cell sorter 100 which provides for sorting of cells or particles in a suspension which are contained in a sample reservoir 102. The suspension is forced into a flow cell chamber 104 where sheathing fluid from a sheathing fluid reservoir 106 surrounds the sample as the sample enters the flow cell 104 from the sample tubing. This combination of sheath fluid and sample focuses the suspension into a serialized order in the resulting stream. The flow cell 104 provides an analysis point where the focused sample intersects a laser beam 108. The differences between the sample and sheath described above are detected in the flow cell 104.
B. Droplet Generator
A droplet generator 114 is also included as a further component of the present invention. The droplet generator 114 perturbs the jet. By doing so, waves of undulations travel down the jet at the velocity of the jet. Preferably, a piezoelectric crystal is utilized to accomplish perturbation of the jet. The frequency of perturbation is set by a frequency generator (not shown), and may be varied as determined by one of skill in the art. The drive amplitude is set by an amplifier (not shown). The jet forms as the stream is forced through the exit nozzle 112 and breaks into droplets at the droplet generator drive frequency.
The elapsed time between the time the sample is detected by the laser 108 in the flow cell 104 to the time that the stream is charged is called the delay time. The delay time must match the transit time of the desired sample from the analysis point to the last attached stream segment 116. The stream configuration must place the last attached stream segment 116 in the same position as the sample to ensure good sorting results. The stream configuration is manipulated in the present invention.
C. Detectors
Another component useful in the present invention is a detection apparatus 118, which monitors the stream for specific particles and provides a characterization of the contents of the stream. Typically, the suspension and the sheath fluid stream 110 typically flow into a cuvette (not shown), which is illuminated by a light source 108. Preferably, the cuvette is present in a visualization chamber or portion of the flow cell 104. However, other chambers may be utilized to contain the suspension to be analyzed and may be selected by one of skill in the art. Suitable light sources include, without limitation, arc lamps, lasers, light bulbs, light emitting diodes (LED), among others. Typically, the light source 108 operates in a continuous mode.
D. Imaging Means
A further component of the present invention includes an imaging means 38 (See FIGS. 6 through 12) to capture an image of the jet below the nozzle 112 according to the present invention. The imaging means can be located in a variety of positions to capture one or more views of the jet, but is preferably located at the droplet forming region 32 (see FIG. 1A) in the performance of this invention. A variety of imaging means are known in the art and can be utilized in the present invention and include the imaging means described herein.
When the optical source or light field 120 illuminates the jet 110 below the nozzle 112, it strobes light at a frequency that is the same as the frequency of the droplet generator 114, e.g., the piezo oscillator, of the flow cytometer 100. The light field 120 can strobe light at the same frequency as the oscillator. In one embodiment, the imaging means is operated at a frequency of about 0.6 to about 100 kilohertz; however the frequency may be adjusted by one of skill in the art as needed.
E. Means for Generating the Numerical Standard and Sample Averages
Typically, the image of the jet (see FIG. 2B) contains noise. Typically, the noise present in the images is generated by the optical illumination source or light field 120. However other components of the instrument can generate undesirable nose in the images. By reducing or eliminating the noise, a more accurate representation of the jet can be obtained. Thus, in order to obtain an accurate image of the jet to be analyzed, the residual noise present in the images of the jet should be eliminated or minimized. Typically, the noise is eliminated by subtracting a background image. The present invention therefore provides for obtaining one or more background images that contain noise (see FIG. 2A). The background images are typically obtained with no sheath or suspension fluid flow present. In order to obtain an accurate background image, multiple images of the background without sheath or suspension fluid flow are obtained. Preferably, about 5 or more background images are obtained. More preferably, about 5 to about 40 background images are obtained. Even more preferably, about 5 to about 10 background images are obtained. Most preferably, about 10 background images are obtained. The multiple background images can optionally be averaged to obtain a background image that is then used according to the invention.
Once the background image and image of the jet are obtained and the same enhanced, the background image is subtracted from the image of the jet to remove noise. See FIG. 2C. If the background image and image of the jet are of the same size, no additional manipulations may be required prior to subtracting the background image from the image of the jet. Conventional and useful noise reduction techniques are also described in the text of Tou and Gonzalez, cited above. If necessary, the images of the jet, before and after background subtraction, can be stored for manual or computer-assisted comparisons at a later date. Alternatively and preferably, the images are instantly obtained and displayed on a monitor. An advantage of the present invention however includes optimizing storage on a computer or optimizing the time required to display on a monitor and instantly processing the images according to the following without storing the same for later use.
G. Adjusting Means
In order to maintain a stable sort, the system can be adjusted prior to and during sorting the particles of the suspension. If a significant deviation of sample average from reference standard is detected, the method involves an adjusting step, in which the processor is programmed to gives commands to the sample pressure regulator, the sheath pressure regulator and/or the piezo oscillator to force the deviant averages back to the reference standard representative of a sort in known, stable and desirable condition. If unable to do so, the processor then issues other commands to the sample pinch, sheath pinch, deflection voltage, stream charge and the user interface to stop the sorting and alerts the user.